Searching for promising sources of grain protectors in extracts from Neotropical Annonaceae

Abstract

To investigate potential sources of novel grain protector compounds against Sitophilus zeamais (Coleoptera: Curculionidae), which is an important insect pest of stored cereals, this study evaluated the bioactivity of ethanolic extracts (66) prepared from 29 species belonging to 11 different genera of Neotropical Annonaceae. A screening assay demonstrated that the most pronounced bioactive effects on S. zeamais were caused by ethanolic extracts from Annona montana, A. mucosa, A. muricata, and A. sylvatica seeds, causing the death of all weevils exposed, almost complete inhibition of the F1 progeny and a drastic reduction in grain losses. Furthermore, the ethanolic extracts obtained from the leaves of A. montana, A. mucosa, A. muricata, and Duguetia lanceolata, especially A. montana and A. mucosa, demonstrated significant bioactive effects on the studied variables; however, the activity levels were less pronounced than in the seed extracts, and the response was dependent on the concentration used. This study is the first to report the activity of secondary metabolites from D. lanceolata on insects as well as the action of A. sylvatica on pests associated with stored grains.

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© 2016
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 15 (4): 215 - 232
ISSN 0717 7917
www.blacpma.usach.cl
Artículo Original | Original Article
215
Searching for promising sources of grain protectors in extracts from
Neotropical Annonaceae
[Búsqueda de fuentes prometedoras de protectores de granos em extractos de Anonaceas Neotropicales]
Leandro do Prado Ribeiro1, José Djair Vendramim1, Gabriel Luiz Padoan Gonçalves1, Thiago Felipe Ansante1,
Eduardo Micotti da Gloria2, Jenifer de Carvalho Lopes3, Renato Mello-Silva3 & João Batista Fernandes4
1Department of Entomology and Acarology and 2Agroindustry, Food and Nutrition Department
University of São Paulo/ “Luiz de Queiroz” College of Agriculture (USP/ESALQ), Piracicaba, São Paulo State, Brazil
3Department of Botany, University of São Paulo (IB/USP), São Paulo, São Paulo State, Brazil
4Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, São Paulo State, Brazil
Contactos | Contacts: Leandro do Prado RIBEIRO - E-mail address: lpribeiro@usp.br
Abstract: To investigate potential sources of novel grain protector compounds against Sitophilus zeamais (Coleoptera: Curculionidae),
which is an important insect pest of stored cereals, this study evaluated the bioactivity of ethanolic extracts (66) prepared from 29 species
belonging to 11 different genera of Neotropical Annonaceae. A screening assay demonstrated that the most pronounced bioactive effects on
S. zeamais were caused by ethanolic extracts from Annona montana, A. mucosa, A. muricata, and A. sylvatica seeds, causing the death of all
weevils exposed, almost complete inhibition of the F1 progeny and a drastic reduction in grain losses. Furthermore, the ethanolic extracts
obtained from the leaves of A. montana, A. mucosa, A. muricata, and Duguetia lanceolata, especially A. montana and A. mucosa,
demonstrated significant bioactive effects on the studied variables; however, the activity levels were less pronounced than in the seed
extracts, and the response was dependent on the concentration used. This study is the first to report the activity of secondary metabolites
from D. lanceolata on insects as well as the action of A. sylvatica on pests associated with stored grains.
Keywords: Allelochemicals, acetogenins, bioactivity, Sitophilus zeamais, stored cereals.
Resumen: Para investigar las posibles fuentes de nuevos compuestos protectores de granos contra Sitophilus zeamais (Coleoptera:
Curculionidae), una importante plaga de los cereales almacenados, este estudio evaluó la bioactividad de los extractos etanólicos (66)
preparados a partir de 29 especies pertenecientes a 11 géneros distintos de Anonaceas Neotropicales. Un ensayo de selección demostró que
los efectos bioactivos más relevantes sobre S. zeamais fueron causados por los extractos etanólicos de las semillas de Annona montana, de
A. mucosa, de A. muricata y de A. sylvatica, que causaron la muerte de todos los gorgojos expuestos, la inhibición parcial de la progenie F1
y una drástica reducción de las pérdidas de grano. Además, los extractos etanólicos obtenidos de las hojas de A. montana, de A. mucosa, de
A. muricata y de Duguetia lanceolata, especialmente de A. montana y de A. mucosa, demostraron efectos bioactivos significativos sobre las
variables estudiadas. Sin embargo, los niveles de bioactividad fueron menores en comparación con los extractos de semillas, y la respuesta
fue dependiente de la concentración utilizada. Este estudio es el primer relato sobre la actividad de los metabolitos secundarios de D.
lanceolata sobre insectos, así como la acción de A. sylvatica sobre plagas asociadas a los granos almacenados..
Palabras clave: Aleloquímicos, acetogeninas, bioactividad, Sitophilus zeamais, cereales almacenados.
Recibido | Received: May 15, 2014
Aceptado | Accepted: May 19, 2015
Aceptado en versión corregida | Accepted in revised form: February 21, 2016
Publicado en línea | Published online: July 30, 2016
Declaración de intereses | Declaration of interests: the São Paulo Research Foundation (FAPESP, grant number 2010/52638-0) and the National Science and Technology Institute
for Biorational Control of Pest Insects (INCT-CBIP, grant number 573742/2008-1) for the financial support..
Este artículo puede ser citado como / This article must be cited as: LP Ribeiro, JD Vendramim, GLP Gonçalves, TF Ansante, EM da Gloria, JC Lopes, R Mello-Silva & JB
Fernandes. 2016. Searching for promising sources of grain protectors in extracts from Neotropical Annonaceae Bol Latinoam Caribe Plant Med Aromat 15 (4): 215 – 232.

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INTRODUCTION
The large diversity of secondary metabolites in plants
(allelochemicals) originates from a long evolutionary
process that relies on the relationships between the
plants and their competitors (natural enemies). Plants
develop these compounds mainly as a defense
mechanism (Wink, 2003). The study of
allelochemicals has not only increased the knowledge
of the processes involved in the interactions between
plants and other factors in the environment but has
also led to the discovery of important bioactive
molecules of great interest for humankind (Ramesha
et al., 2011). Among other functions, these bioactive
molecules are used as model-prototypes for the
development of new drugs (Miller, 2011) and new
products for the protection of agricultural crops and
stored commodities (Cantrell et al., 2012).
The structural and functional diversity of
allelochemicals is a key factor for the survival and
evolutionary success of plant species inhabiting an
environment with an abundance of natural enemies.
Therefore, the tropical flora, with its unique
biodiversity, is a promising natural reservoir of
bioactive substances (Valli et al., 2012). In this
context, Brazil exhibits enormous potential for the
development of novel active substances based on
natural products because the country has the highest
plant genetic diversity in the world, with more than
55,000 catalogued species (Simões & Schenkel,
2002). However, to date, this potential has not been
well exploited.
Among the botanical families that occur in
the Neotropical regions, Annonaceae is the main
family of the order Magnoliales (APG III, 2009) and
is one of the most specious families of angiosperms
comprising 135 genera and approximately 2,500
species (Chatrou et al., 2004). Annonaceae exhibits a
pantropical distribution with 40 genera and 900
species in the Neotropical region. In Brazil, this
family is represented by 29 genera, of which 1 are
endemic, and 386 species, and a large proportion of
this richness is found in the Amazon Rain Forest and
Atlantic Forest (Maas et al., 2013).
Despite the lack of studies, a large number of
diverse chemical compounds present in the different
structures of Annonaceae plants have been isolated.
Alkaloids, acetogenins, diterpenes and flavonoids are
the main chemical groups in extracts from the bark,
branches, leaves, fruits and seeds of Annonaceae
(Lebouef et al., 1982; Chang et al., 1998; Kotkar et
al., 2001). Among these classes, acetogenins are
conspicuous because of the vast array of biological
activities they exhibit. Acetogenins are a series of
natural products (C-35/C-37) derived from long-
chain fatty acids (C-32/C-34) combined with a 2-
propanol unit (Alali et al., 1999).
Our previous studies (Ribeiro, 2010; Ribeiro
et al., 2013) demonstrated a promising grain-
protectant effect in seed extracts from two species of
Annona, which are characterized by a complex
mixture of acetogenins and alkaloids. This
observation motivated additional biomonitoring
investigations in other Annonaceae species in order
to explore more comprehensively the richness of
allelochemicals in this plant family and the species
diversity of the Brazilian flora. These studies
prompted our research program to search for
allelochemicals with activities against pest species of
stored grains, which is an essential component of
current stored grain integrated pest management
programs (IPM).
This study evaluated the bioactivity of
ethanolic extracts (66) of different structures from 29
Annonaceae species (7.5% of all Brazilian species)
belonging to 11 different genera (Anaxagorea,
Annona, Duguetia, Ephedranthus, Guatteria,
Hornschuchia, Oxandra, Porcelia, Pseudoxandra,
Unonopsis, and Xylopia) against Sitophilus zeamais
Motschulsky (Coleoptera: Curculionidae), which is
an important pest of stored cereals under tropical
conditions. In addition, the fungicidal and
antiaflatoxigenic activities of the promising extracts
were evaluated in vitro against the isolate CCT7638
of Aspergillus flavus Link (Ascomycota: Eurotiales:
Trichocomaceae), a producer of aflatoxin B1, in order
to better characterize the potential of these extracts as
grain protectors.
MATERIAL AND METHODS
Species sampling and plant extract preparation
The collection data for the plant species used in the
study, which were obtained from different locations in
the south and southeast regions of Brazil, are shown in
Table 1. In total, 29 species of Annonaceae belonging
to 11 genera were collected and were investigated.

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For the extract preparation, the plant structures
were collected and were dehydrated in an oven at 40º
C for 48 to 72 hours. Subsequently, the materials
were separately milled in a knife mill to obtain a
powder of each plant structure, which were stored
separately in sealed glass containers until use. The
organic extracts were obtained by maceration in
ethanol solvent (1:5, w v-1). For this step, the plant
powder was maintained in the solvent for 3 days after
which it was filtered through filter paper. The
remaining residual cake was placed back in the
ethanol solvent, and this process was repeated 4
times. The solvent remaining in the filtered solution
was eliminated in a rotary evaporator at 50º C and -
600 mm Hg pressure. After the solvent was
evaporated in the airflow chamber, the extraction
yield of each structure for all species was determined.
Bioassays
The bioassays were performed in a climate-controlled
room at 25 ± 2º C, 60 ± 10% relative humidity, a
photoperiod of 14 hours and a mean luminosity of
200 lux. Whole corn grains were used as the substrate
for the assays. The corn grains were manually
selected from the hybrid AG 1051 (yellow dent,
semi-hard) from crops developed without
insecticides. The experimental design used for the
tests was completely randomized.
A microatomizer coupled to a pneumatic
pump and adjusted to a pressure of 0.5 kgf cm-2 with
a spray volume of 30 L t-1 was used for the
application of the treatments. After spraying, the
grains/extract mixture was manually placed in 2-liter
plastic bags, which were lightly shaken for 1 minute.
Screening for the identification of promising
extracts
To identify extracts with bioactivity against S.
zeamais, bioassays were performed to verify the
insecticidal activity and sublethal effects, which were
assessed by evaluating the number of insects emerged
(F1 progeny) and the damage to the treated samples.
As a large number of extracts was obtained, they
were divided into 5 groups in order to be tested. The
groups were established according to the similarity of
collection dates and plant structures available for
each species. The extracts were assayed at
concentration of 3,000 mg kg-1 (mg of extract kg-1
of corn), which were defined based on previous
studies (Ribeiro, 2010).
Evaluation of insecticidal activity
For this bioassay, corn samples (10 g, in Petri dishes
measuring 6-cm in diameter × 2-cm high) were
treated separately with the respective extracts. The
growth substrate treated with the solvent
[acetone:methanol solution (1:1, v v-1)] was used as
the control. Preliminary assays were performed to
evaluate the possible effects of the solution used for
the resuspension of the extracts on S. zeamais. Next,
each Petri dish was infested with 20 adult S. zeamais
(aged 10 to 20 days) from both sexes, and 10
replicates per treatment were performed. The adult
survival was evaluated on day 10 after infestation.
The insect was considered dead when its extremities
were completely distended and it exhibited no
reaction to contact with a paintbrush for 1 minute.
Evaluation of F1 progeny and damages
The same sampling units used for the insecticidal
assay were used in this bioassay. The grains were
treated with the respective extracts and were infested
with 20 adults from both sexes (aged 10 to 20 days).
After 10 days of infestation, the adults were removed,
and the sampling units were kept under the climate
conditions previously described. As before, 10
replicates per treatment were performed.
At 60 days after the initial infestation, the
number of emerged adults in each dish was counted.
The damages caused by the feeding of the S. zeamais
were determined through the visual verification of the
percentage of damaged or perforated grains in each
sample. In addition, the grain weight loss (%) was
estimated based on the equation proposed by Adams
and Schulten (1976).

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Table 1
Species and structures of Annonaceae used in the study and the respective collection data.
Species
Plant structures
Local of collection
Data of
collection
Voucher
(Herbarium)1
Anaxagorea dolichocarpa
Sprague & Sandwith
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º08’04,0” S; 40º03’24,5” W;
elevation: 33 m)
17/11/2011
Lopes 361
(CVRD, ESA, SPF)
Annona acutiflora
Mart.
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º09’05,4” S; 40º04’02,4” W;
elevation: 70 m)
16/11/2011
Lopes 144
(CVRD, ESA, SPF)
Annona cacans
Warm.
Leaves, branches,
and seeds
IAC/APTA, Jundiaí, SP, Brazil
(23º06’56,6” S; 46º55’58,3” W;
elevation: 599 m)
04/03/2011
Ribeiro 17
(ESA)
Annona dolabripetala
Raddi
Leaves and
branches
Botanical Garden of São Paulo, São
Paulo, SP, Brazil
(23º32’18,2” S; 46º36’44,5” W;
elevation: 745 m)
07/06/2011
Ribeiro 18
(ESA)
Annona emarginata
(Schltdl.) H.Rainer
Leaves and
branches
IAC/APTA, Jundiaí, SP, Brazil
(23º06’53,4” S; 46º56’07,2” W;
elevation: 616 m)
04/03/2011
Ribeiro 16
(ESA)
Annona montana
Macfad.
Leaves, branches,
and seeds
ESALQ/USP Campus, Piracicaba,
SP, Brazil
(22º42’28,2” S; 47º37’59.4” W;
elevation: 537 m)
21/03/2011
Ribeiro 3
(ESA)
Annona mucosa
Jacq.
Leaves, branches,
and seeds
ESALQ/USP Campus, Piracicaba,
SP, Brazil
(22º42’28,5” S; 47º37’59.6” W;
elevation: 534 m)
17/03/2011
Ribeiro 4
(ESA)
Annona muricata
L.
Leaves, branches,
and seeds
ESALQ/USP Campus, Piracicaba,
SP, Brazil
(22º42’25,4” S; 47º37’43,9” W;
elevation: 576 m)
12/04/2011
Ribeiro 12
(ESA)
Annona reticulata
L.
Leaves and
branches
ESALQ/USP Campus, Piracicaba,
SP, Brazil
(22º42’51,4” S; 47º37’38,8” W;
elevation: 548 m)
01/03/2011
Ribeiro 11
(ESA)
Annona sp. 1
Leaves and
branches
São Luís Farmer, Descalvado, SP,
Brazil
(21⁰52’58,0” S; 47⁰40’38,0” W;
elevation: 679 m)
02/04/2011
Ribeiro 13
(ESA)
Annona sp. 2
Leaves and
branches
Frutas Raras Farmer, Rio Claro, SP,
Brazil
(23º06’53,4” S; 46º56’07,2” W;
elevation: 716 m)
02/04/2011
Ribeiro 14
(ESA)
Annona sylvatica
A.St.-Hil.
Leaves, branches,
and seeds
Ribeiro Small Farmer, Erval Seco,
RS, Brazil
(27º25’41,8” S; 53º34’11,2” W;
elevation: 466 m)
25/04/2011
Ribeiro 10
(ESA)
Duguetia lanceolata
A.St. Hil.
Leaves, branches,
and seeds
ESALQ/USP Campus, Piracicaba,
SP, Brazil
(22º42’41,5” S; 47º38’0,2” W;
elevation: 556 m)
23/03/2011
Ribeiro 9
(ESA)
Ephedranthus dimerus J.C.Lopes, Chatrou
& Mello-Silva (1)
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º09’05,5” S; 40º04’00,1” W;
elevation: 67 m)
17/11/2011
Lopes 145
(CVRD, ESA, SPF, WAG)

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Ephedranthus dimerus J.C.Lopes, Chatrou
& Mello-Silva (2)
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º10’19,2” S; 40º01’08,7” W;
elevation: 48 m)
17/11/2011
Lopes 362
(CVRD, ESA, SPF, WAG)
Guatteria australis
A. St.-Hil.
Leaves and
branches
Vale Natural Reserve,Linhares, ES,
Brazil
(19º08’28,5” S; 40º04’05,7” W;
elevation: 18 m)
17/11/2011
Lopes 153
(CVRD, ESA, SPF)
Guatteria ferruginea
A. St.-Hil.
Leaves and
branches
Santa Lúcia Biological Station, Santa
Tereza, ES, Brazil
(19º58’02,5” S; 40º32’15,5” W;
elevation: 694 m)
13/11/2011
Lopes 348
(ESA)
Guatteria sellowiana
Schltdl.
Leaves and
branches
Santa Lúcia Biological Station, Santa
Tereza, ES, Brazil
(19º58’02,5” S; 40º32’15,5” W;
elevation: 694 m)
13/11/2011
Lopes 345
(ESA)
Guatteria villosissima
A. St.-Hil.
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º09’25,6” S; 40º31’43,6” W;
elevation: 636 m)
16/11/2011
Lopes 146
(ESA)
Hornschuchia bryotrophe Nees
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º07’56,4” S; 40º05’05,7” W;
elevation: 58 m)
17/11/2011
Lopes 111
(ESA)
Hornschuchia citriodora
D.M. Johnson
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º07’57,8” S; 40º05’05,9” W;
elevation: 48 m)
17/11/2011
Lopes 110
(ESA)
Hornschuchia myrtillus
Nees
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º11’13,3” S; 39º54’49,6” W;
elevation: 44 m)
17/11/2011
Lopes 364
(CVRD)
Oxandra martiana
(Schltdl.) R E. Fr.
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º11’30,8” S; 39º57’09,3” W;
elevation: 31 m)
17/11/2011
Lopes 363
(CVRD, ESA, SPF)
Porcelia macrocarpa
(Warm.) R.E. Fries
Leaves and
branches
Botanical Garden of São Paulo, São
Paulo, SP, Brazil
(23º33’55,8” S; 46º36’08,3” W;
elevation: 752 m)
07/06/2011
Eiten 791
(SP)
Pseudoxandra spiritus-sancti
Maas
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º04’44,8” S; 39º53’19,5” W;
elevation: 20 m)
16/11/2011
Lopes 317
(CVRD, SPF)
Unonopsis sanctae-teresae
Maas & Westra
Leaves and
branches
Goiapaba-açu Road, Santa Teresa,
ES, Brazil
(19º54’50,5” S; 40º31’59,1” W;
elevation: 840 m)
14/11/2011
Lopes 355
(ESA)
Xylopia brasiliensis
Spreng.
Leaves and
branches
Augusto Ruschi Biological Reserve,
Santa Tereza, ES, Brazil
(19º54’27,1” S; 40º33’02,0” W;
elevation: 805 m)
14/11/2011
Lopes 351
(ESA)
Xylopia decorticans
D.M. Johnson & Lobão
Leaves and
branches
Augusto Ruschi Biological Reserve,
Santa Tereza, ES, Brazil
(19º54’28,5” S; 40º32’57,3” W;
elevation: 807 m)
14/11/2011
Lopes 352
(ESA)
Xylopia frutescens
Aubl.
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
16/11/2011
Lopes 359
(ESA)

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(19º09’23,4” S; 39º59’30,3” W;
elevation: 30 m)
Xylopia laevigata
(Mart.) R.E. Fr.
Leaves and
branches
Vale Natural Reserve, Linhares, ES,
Brazil
(19º05’01,2” S; 39º53’04,8” W;
elevation: 22 m)
16/11/2011
Lopes 316
(ESA)
1 CVRD (Vale Natural Reserve Herbarium, Linhares, ES, Brazil); ESA (“Luiz de Queiroz” College of Agriculture
Herbarium, Piracicaba, SP, Brazil); SP (Botanical Institut of São Paulo Herbarium, São Paulo, Brazil); SPF
(University of São Paulo Herbarium, São Paulo, SP, Brazil); WAG (Wageningen University Herbarium,
Wageningen, Netherlands).
Concentration-response curves of active extracts
The extracts demonstrating the most promising
results were bioassayed for the estimation of the LC50
and LC90, corresponding to the concentration
necessary to kill 50% and 90% of the population of
weevils, respectively. For these estimations,
preliminary tests were performed to determine the
baseline concentrations that caused the death of 95%
of the adults and a mortality rate similar to that in the
control. Based on these results, the test
concentrations (5-7 concentrations; range: 50 - 4,000
mg kg-1) were established using the formula
proposed by Finney (1971). The remaining
experimental procedures were the same as those used
in the initial screening, in which the mortality was
assessed 10 days after the infestation of the sample
units.
Estimation of average lethal time (LT50) of the
promising extracts
For each selected extract, the time required to kill
50% of the weevil population (LT50) was estimated
based on the LC90 value determined in the previous
bioassay. The same procedures described in the
screening assay were used for this bioassay; however,
the evaluation of weevil mortality was performed
every 24 hours for 10 days.
Fungicidal and antiaflatoxigenic effects of the most
promising extracts
The antifungal and antiaflatoxigenic activities
(against isolate CCT7638 of A. flavus, a producer of
AFB1) of the most promising extracts were evaluated
using a method termed poison food (Alvarez-
Castellanos et al., 2001). This technique is based on
the observation of the growth of fungal mycelium in
YES (yeast extract saccharose) culture media using
1,000 mg L-1 of the respective extracts (final
concentration) dissolved in 5 mL solvent solution
[acetone: water, (1:3, v v-1)], incorporated by manual
agitation in unfused culture media (temperature
approximately 45º C). The extract + medium (10 mL)
were added to each Petri dish (6.5-cm diameter), and
10 dishes were used per treatment. The solvent
solution (acetone: water) was included as a control,
and water was used as the negative control.
The fungus was inoculated following the
solidification of the media as follows: the central area
of the Petri dish was perforated using a Stanley knife
previously immersed in a conidia solution. After
incubation of the fungal colonies for 11 days in PDA
(potato, dextrose and agar) media, the spore
suspension was prepared by scraping the media
surface using a Drigalski spatula followed by
immersion in 50 mL of an aqueous solution (47.5 mL
of distilled water + 2.5 mL of dimethyl sulfoxide).
The amount of conidia in the solution was
standardized to contain 2 to 9 × 105 conidia mL-1,
measured using a Neubauer chamber. The inoculated
dishes were sealed with plastic film and were
incubated upside down at 25 ± 2º C and a scotophase
of 24 hours.
The radial mycelial growth was evaluated at
48, 96, 144 and 192 hours after the fungal
inoculation. The evaluation consisted of measuring
the diameter of each colony in 2 directions at a
straight angle using a caliper. Based on the arithmetic
mean of the 2 measurements, the percentage
inhibition (P.I.) of the treatments relative to the
control was calculated using the following equation:

100/)(.. ControlTreatmentControlIP 
The production of aflatoxin (B1) by isolate
CCT7638 of A. flavus grown in culture media
containing the respective treatments was assessed

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using the thin-layer chromatography (TLC)
technique. The culture media from 5 Petri dishes
randomly chosen from each treatment was transferred
to a 50-mL Falcon tube using Stanley knives and
spatulas. The weight of the transferred material was
recorded, and the material was subjected to an
aflatoxin extraction process.
For the extraction of aflatoxins from the
media, 14 mL of distilled water and 18 mL of
analysis-grade methanol were added to the tube. The
content was vortexed for 1 minute. A 5-mL aliquot of
the extract was transferred to an amber vial, and the
solution volume was evaporated entirely using a
sample concentrator with airflow at 45º C. The dried
material was redissolved in 200 μL of toluene:
acetonitrile (9:1) and agitated for 30 seconds using
ultrasound. The presence and quantification of
aflatoxins in the extract were performed in aluminum
chromatoplates with silica gel. The development of
the chromatography plates was performed in vats
containing 5 mL of the elution solution composed of
ether:methanol:water (96:3:1). The presence of
aflatoxins in the samples was verified by comparison
to the AFB1 standard. The standard AFB1 solution
was prepared based on the Sigma-Aldrich standard
(Sigma AF-1), and the concentration was determined
according to methodology 971.22 found in the
American Official Analytical Chemistry (2006). For
this assay, the concentration of aflatoxins in the
samples was compared with 3-, 4- and 6-µL aliquots
of the AFB1 standard. The calculation of
contamination was performed according to the
following equation:
Where: AF= aflatoxin content (AFB1); Y =
standard concentration in μg mL-1; S = μL of the
standard toxin with fluorescence equivalent to the
sample; V = extract final volume (sample) in μL; X =
extract initial volume (sample) in μL; W = sample
weight, in grams, in the final extract.
Data analysis
Generalized linear models (GLM) (Nelder &
Wedderburn, 1972) with quasi-binomial distributions
were used for the analysis of the proportions of
mortality and damaged grains, whereas GLM with
quasi-Poisson distributions was used for the analysis
of emerged insect numbers. GLM with Gaussian
distribution was used for the analysis of A. flavus
vegetative growth and aflatoxin production. In all
cases, the goodness-of-fit was determined using a
half-normal probability plot with a simulated
envelope (Hinde & Demétrio, 1998). When a
significant difference was observed between the
treatments, multiples comparisons (Tukey’s test, P <
0.05) were performed using the glht function of the
multicomp package with adjustment of P values.
Multivariate analyses were performed to
determine the grouping of the crude extracts of
Annonaceae based on the variables analyzed in the
screening assay. The mean Euclidian distance was
used as a measurement of similarity, and the
UPGMA (unweighted pair-group average) method
was used as a clustering strategy. The relationship
between the variables analyzed was determined using
Spearman’s nonparametric analysis (P = 0.05). The
analyses were performed using the software “R”,
version 2.15.1 (R Development Core Team, 2012).
A binomial model with a complementary
log-log link function (gompit model) was used to
estimate the lethal concentrations (LC50 and LC90),
using the Probit Procedure in the software SAS
version 9.2 (SAS Institute, 2011). Finally, the mean
lethal time (LT50) was estimated using the method
proposed by Throne et al. (1995) for Probit analysis
of correlated data.
RESULTS
Selection of the promising crude extracts
Of the 66 tested extracts at 3,000 mg kg-1, the most
pronounced bioactive effects on S. zeamais were
caused by the ethanolic extracts from the A. montana,
A. mucosa, A. muricata and A. sylvatica seeds (Table
2). These extracts produced complete mortality of the
exposed weevils, almost total inhibition of the F1
progeny and a drastic reduction in damage to the
treated samples. The ethanolic extracts from the A.
montana, A. mucosa, A. muricata and D. lanceolata
leaves, especially the A. montana and A. mucosa
leaves, exhibited significant bioactive effects;
however, compared with the seed extracts, the effects
were at lower levels and exhibited a concentration-
dependent response.
 
)/()( XWYSVAF 

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/222
Table 2
Mortality (mean ± standard error) on day 10, number of emerged insects (F1 progeny) and damage after 60
days of infestation with Sitophilus zeamais (Coleoptera: Curculionidae) in corn samples (10 g) treated with
ethanolic extracts from different species and/or structures of Annonaceae (3,000 mg kg-1)*.
Species
Plant
structures
Mortality
(%)1
No. of emerged
insects2
% Grains
damaged1
Grain weight losses
Total (%)3
Relative (%)4
Group A[2]
Annona cacans
Leaves
0.50±0.50 c
51.00±5.12 a
86.98±6.99 a
10.87±0.87
96.71
Branches
0.00±0.00
54.20±2.64 a
89.09±1.54 a
11.13±0.19
99.02
Seeds
14.50±2.03 c
41.50±4.11 ab
76.20±3.01 a
9.52±0.37
84.70
Annona montana
Leaves
77.50±5.73 a
8.40±3.35 cd
17.71±6.90 c
2.21±0.86
19.66
Branches
1.50±0.76 c
46.90±2.24 a
82.01±2.57 a
10.25±0.32
91.19
Seeds
100.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00
0.00
Annona mucosa
Leaves
83.00±5.33 a
6.10±1.79 d
13.54±3.84 c
1.69±0.48
15.04
Branches
0.00±0.00
59.90±3.92 a
92.22±2.68 a
11.52±0.33
102.49
Seeds
100.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00
0.00
Annona muricata
Leaves
34.00±6.27 b
25.90±4.61 bc
48.31±7.50 b
6.03±0.93
53.65
Branches
1.50±0.76 c
47.40±2.74 a
86.38±2.81 a
10.79±0.35
96.00
Seeds
100.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00
0.00
Annona sylvatica
Leaves
19.50±4.18 b
41.40±4.32 ab
74.67±4.49 ab
9.36±0.56
83.27
Branches
0.00±0.00
46.70±2.84 a
83.39±3.30 a
10.42±0.41
92.70
Seeds
100.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00
0.00
Duguetia lanceolata
Leaves
37.50±4.60 b
17.00±2.74 c
42.35±6.13 b
5.29±0.76
47.06
Branches
1.00±0.66 c
51.04±2.71 a
87.58±2.21 a
10.94±0.27
97.33
Seeds
0.50±0.50 c
51.00±2.40 a
89.23±1.93 a
11.15±0.24
99.20
Control
(acetone:methanol, 1:1
(v/v))
--
0.00±0.00
51.40±3.72 a
89.97±3.10 a
11.24±0.38
--
F
57.24
24.24
25.94
P value
< 0.0001
< 0.0001
< 0.0001
Group B[2]
Annona dolabripetala
Leaves
4.50±1.74 ab
28.50±2.70 c
61.90±3.74 d
7.73±0.46
70.98
Branches
0.00±0.00
57.80±2.07 ab
88.44±2.41 abc
11.05±0.30
101.47
Annona emarginata
Leaves
0.00±0.00
46.90±2.79 b
84.49±3.65 abc
10.56±0.45
96.97
Branches
0.00±0.00
55.1±1.64 ab
95.10±0.48 a
11.88±0.06
109.09
Annona reticulata
Leaves
5.00±2.23 ab
43.10±3.98 b
82.08±4.19 abc
10.26±0.52
94.21
Branches
3.00±1.10 ab
44.50±4.43 b
79.71±6.60 c
9.96±0.82
91.46
Annona sp.1
Leaves
0.50±0.50 b
47.30±3.26 b
81.03±3.23 bc
10.12±0.40
92.93
Branches
0.00±0.00
68.40±3.47 a
94.35±1.10 abc
11.79±0.13
108.26
Annona sp. 2
Leaves
16.50±4.15 a
27.90±3.32 c
58.57±4.56 d
7.32±0.57
67.22
Branches
0.00±0.00
54.80±2.41 ab
88.75±1.63 abc
11.09±0.20
101.84
Porcelia macrocarpa
Leaves
0.00±0.00
68.30±1.66 a
94.72±1.31 ab
11.84±0.16
108.72
Branches
0.50±0.50 b
53.20±3.00 ab
91.78±1.80 abc
11.47±0.22
105.33
Control
(acetone:methanol, 1:1
(v/v))
--
2.00±1.52 ab
51.00±2.48 b
87.17±2.64 abc
10.89±0.33
--
F
7.23
16.29
12.38
P value
<0.0001
<0.0001
<0.0001

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/223
Group C[2]
Annona acutiflora
Leaves
7.00±2.26
27.70±2.57
65.84±3.45
8.23±0.43
94.93
Branches
4.50±1.38
37.50±3.27
81.37±3.97
10.17±0.49
117.30
Guatteria australis
Leaves
6.00±2.76
32.30±3.50
74.80±3.58
9.35±0.44
107.84
Branches
6.50±1.50
32.90±3.90
72.72±4.75
9.09±0.59
104.84
Guatteria ferruginea
Leaves
9.00±2.66
25.00±3.09
62.16±3.28
7.77±0.41
89.62
Branches
8.50±2.24
30.00±1.61
74.67±3.30
9.33±0.41
107.61
Guatteria sellowiana
Leaves
7.50±3.00
33.90±4.37
73.82±6.04
9.22±0.75
106.34
Branches
2.00±0.81
35.70±2.64
78.21±2.73
9.77±0.34
112.69
Guatteria villosissima
Leaves
3.50±1.50
30.50±3.00
69.52±5.72
8.69±0.71
100.23
Branches
2.00±1.10
38.20±2.45
77.57±2.81
9.69±0.35
111.76
Unonopsis sanctae-
teresae
Leaves
4.00±1.45
32.70±2.13
74.51±3.49
9.31±0.43
107.38
Branches
3.00±1.10
28.60±2.85
67.41±5.18
8.42±0.64
97.12
Control
(acetone:methanol, 1:1
(v/v))
--
3.00±1.33
29.00±2.60
69.37±5.17
8.67±0.64
100.00
F
1.81ns
1.66 ns
1.65 ns
P value
0.0540
0.0834
0.0868
Group D[2]
Oxandra martiana
Leaves
0.00±0.00
46.80±2.96 ab
87.98±3.45
10.99±0.43
101.85
Branches
2.50±0.83
45.20±4.18 ab
85.78±3.58
10.72±0.44
99.35
Pseudoxandra spiritus-
sancti
Leaves
3.00±1.52
38.00±1.69 b
83.21±2.14
10.40±0.26
96.39
Branches
4.00±2.33
41.90±3.91ab
80.60±5.03
10.07±0.62
93.33
Xylopia brasiliensis
Leaves
0.00±0.00
48.70±2.72 ab
90.20±2.94
11.27±0.36
104.45
Branches
1.00±0.66
49.80±2.48 ab
90.01±1.74
11.25±0.21
104.26
Xylopia decorticans
Leaves
0.50±0.50
44.30±2.81 ab
86.21±2.79
10.77±0.34
99.81
Branches
0.50±0.50
41.80±2.90 ab
86.16±2.79
10.77±0.34
99.81
Xylopia frutescens
Leaves
0.00±0.00
44.20±4.76 ab
83.65±4.44
10.45±0.55
96.85
Branches
0.50±0.50
56.40±4.83 a
89.72±3.66
11.21±0.45
103.89
Xylopia laevigata
Leaves
1.00±0.66
38.50±1.63 b
81.53±2.50
10.19±0.31
94.44
Branches
0.50±0.50
45.60±2.71 ab
88.24±2.48
11.03±0.31
102.22
Control
(acetone:methanol, 1:1
(v/v))
--
0.50±0.50
41.50±2.73 ab
86.35±2.85
10.79±0.35
100.00
F
1.78 ns
2.31
0.94 ns
P value
0.8097
0.0110
0.5079
Group E[2]
Anaxagorea
dolichocarpa
Leaves
9.50±3.11 a
35.60±3.46 ab
75.23±3.50 ab
9.40±0.43
97.11
Branches
4.50±1.74 a
47.60±4.59 a
85.66±3.35 ab
10.70±0.41
110.54
Ephedranthus dimerus
1
Leaves
1.50±1.06 a
39.50±4.48 ab
77.55±6.33 ab
9.69±0.79
100.10
Branches
0.50±0.50 a
34.40±3.21 ab
79.98±4.41 ab
9.99±0.55
103.20
Ephedranthus dimerus
2
Leaves
8.50±2.98 a
32.40±4.90 ab
71.11±8.13 ab
8.88±1.01
91.74
Branches
1.00±0.66 a
45.50±2.68 a
89.06±2.52 a
11.13±0.31
114.98
Hornschuchia
bryotrophe
Leaves
7.50±2.81 a
23.80±2.36 b
60.89±5.75 b
7.61±0.71
78.62
Branches
3.00±0.81 a
38.80±4.92 ab
77.94±5.48 ab
9.74±0.68
100.62
Hornschuchia
citriodora
Leaves
8.00±3.81 a
44.00±4.48 a
85.62±4.46 ab
10.70±0.55
110.54
Branches
1.00±0.66 a
34.60±4.07 ab
74.66±4.40 ab
9.33±0.55
96.38
Hornschuchia myrtillus
Leaves
12.00±5.22 a
27.88±4.96 ab
62.60±6.92 b
7.82±0.66
80.79

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/224
Branches
2.00±1.10 a
48.80±2.96 a
87.45±2.07 a
10.93±.025
112.91
Control
(acetone:methanol, 1:1
(v/v))
--
1.00±0.66 a
37.40±5.89 ab
77.48±6.17 ab
9.68±0.77
100.00
F
3.72
3.09
2.84
P value
0.0115
0.0108
0.0190
1Means followed by different letters in the columns containing each tested group extracts indicate
significant differences between treatments (GLM with quasi-binomial distribution followed by Tukey’s post
hoc test, P<0.05); 2Means followed by different letters in the columns containing each tested group extracts
indicate significant differences between treatments (GLM with quasi-Poisson distribution followed by
Tukey’s post hoc test, P<0.05); 3Calculated using the formula proposed by Adams and Schulten (1976);
4Calculated based on the relative comparison of the treatment (extract) with its respective control;
*Applied using a spray volume of 30 L t-1; ns: Not significant (P>0.05)
The hierarchical grouping analysis using the
data from the variables analyzed in the screening
bioassays indicated the formation of 3 groups (Figure
1). The first group comprised the ethanolic extracts
from the A. montana, A. mucosa, A. muricata and A.
sylvatica seeds, as well as extracts from the A.
montana and A. mucosa leaves, which demonstrated
the most pronounced lethal and sublethal effects. The
second group comprised the ethanolic extracts from
D. lanceolata and A. muricata leaves, species that
demonstrated less pronounced bioactive effects
(lethal and sublethal). The third group encompassed
the controls and extracts that did not demonstrate
bioactivity against the targeted pest.
Independently of the concentration tested, the
adult mortality was inversely correlated with the
other tested variables [F1 progeny (r = -0.59; P <
0.0001) and % damaged grains(r = -0.58; P <
0.0001)]. Although mortality was the variable with
the highest weight in the separation between
treatments, based on the Spearman’s correlation
coefficients, one cannot discard the small
oviposition- and/or feeding-deterrent action of the
extracts from the respective Annonaceae species.
Estimation of lethal concentrations and average
lethal time of the selected extracts
The extract prepared from the A. mucosa seeds
demonstrated the lowest LC50 and LC90 values
(288.33 and 505.47 mg kg-1, respectively) (Table 3).
These values were significantly different from the
values for the other active extracts based on the
comparison of the estimated confidence intervals (P
< 0.05). In general, the extracts from the seeds of
different bioactive Annona species demonstrated the
lowest values compared with the active extracts from
leaves. On the other hand, the average lethal time
(varying between 82.06 and 94.85 hours) did not
demonstrate great differences between the treatments
(Table 4), showing a slower activity of the active
extracts, which is probably related with the
mechanisms of action of the active compounds.
Fungicidal and antiaflatoxigenic activity of the most
promising extracts
Overall, the ethanolic extracts tested (1,000 mg kg-1),
which were selected based their activity on S.
zeamais, did not significantly inhibit the vegetative
growth of the isolate CCT7638 of A. flavus and did
not affect the production of AFB1 after 192 hours of
incubation (Table 5). However, the extract from the
A. sylvatica seeds reduced the initial growth rate
(fungistatic effect) of the radial mycelial growth after
48 hours of incubation. Based on the results, the
toxicity of the solvent used for extract solubilization
(acetone) in A. flavus was verified. Although the
concentration was low (25%), caution is
recommended when using this organic solvent in
these types of bioassays.

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/225
Figure 1
Dendrogram obtained from Cluster analysis based on bioactivity similarity of ethanolic extracts from
Annonaceae on Sitophilus zeamais (Coleoptera: Curculionidae) [mean Euclidian distance as dissimilarity
measurement and UPGMA (Unweighted Pair Group Method) as a clustering strategy method]
at 3,000 mg kg-1.

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/226
Table 3
Estimation of LC50 and LC90 (in mg kg-1*) and confidence interval of ethanolic extracts from Annonaceae
for Sitophilus zeamais (Coleoptera: Curculionidae) adults after 10 days of exposure in treated maize
samples (10 g).
Species
(structures)
n 1
Slope ± SE
(valor de P)
LC50
(CI) 2
LC90
(CI) 2
χ2 (3)
d.f. 4
h.5
Annona montana
(leaves)
1,400
4.86±0.29
(P<0.0001)
1,851.00
(1,758.00 –1,942.00)
3,270.00
(3,075.00 –3,516.00)
3.75
4
0.94
Annona montana
(seeds)
1,200
7.09±0.42
(P<0.0001)
621.70
(557.11–677.44)
942.45
(858.96–1,071.91)
5.55
3
1.85
Annona mucosa
(leaves)
1,400
5.74±0.46
(P<0.0001)
1,972.00
(1,847.00–2,080.00)
3,190.00
(3,026.00–3,405.00)
1.56
4
0.82
Annona mucosa
(seeds)
1,600
4.92±0.33
(P <0.0001)
288.33
(267.29–307.21)
505.47
(478.58–537.75)
4.71
5
0.94
Annona muricata
(seeds)
1,600
6.35±0.37
(P <0.0001)
384.94
(364.47–403.75)
594.76
(569.35–624.19)
4.88
5
0.98
Annona muricata
(leaves)
--
--
>3,000
--
--
--
--
Annona sylvatica
(seeds)
1,200
6.32±0.75
(P <0.0001)
554.48
(471.11–617.90)
858.58
(799.49–918.24)
3.07
3
0.37
Duguetia lanceolata
(leaves)
--
--
>3,000
--
--
--
--
1 n: number of tested insects; 2 CI: 95% confidence interval; 3 χ2: calculated chi-squared value;
4 d.f.: degrees of freedom; 5 h.: heterogeneity factor; * Applied using a spray volume of 30 L t-1;
-- Not determined.
Table 4
Estimation of average lethal time (LT50, in hours) and confidence interval of ethanolic extracts from
Annonaceae for Sitophilus zeamais (Coleoptera: Curculionidae) adults.
Species
(structures)
n 1
Slope ± SE
LT50 (CI) 2
χ2 (3)
d.f. 4
h.5
Annona montana
(leaves)
400
8.30±0.41
88.76
(83.39–94.34)
18.19
7
2.60
Annona montana
(seeds)
400
7.08±0.30
86.67
(82.12–91.08)
15.39
8
1.98
Annona mucosa
(leaves)
400
5.98±0.24
94.85
(88.89–100.68 )
13.67
7
1.95
Annona mucosa
(seeds)
400
6.31±0.18
86.13
(84.04–88.17)
7.60
8
0.95
Annona muricata
(seeds)
400
5.61±0.22
90.42
(84.32–96.23)
19.14
8
2.39
Annona sylvatica
(seeds)
400
7.18±0.23
82.06
(78.81–85.21)
16.69
8
2.09
1 n: number of tested insects; 2 CI: 95% confidence interval;
3 χ2: calculated chi-squared value; 4 d.f.: degrees of freedom;
5 h.: heterogeneity factor.

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/227
Table 5
Radial growth (mean ± standard error) of isolate CCT7638 of Aspergillus flavus colonies and production of
aflatoxin (AFB1) in YES (yeast extract saccharose) culture media containing ethanolic seed extracts from
different Annonaceae (1,000 mg L-1).
Extracts / Incubation time
Diameter of colonies (mm) 1
Production of
48 hours
P.I.
(%)2
96 hours
P.I.
(%)2
144 hours
P.I.
(%)2
192 hours
P.I.
(%)2
AFB1 (ppm mm-
2) 1
Annona montana
10.30±0.88
b
+27.16
37.90±2.02
b
+22.65
50.80±2.09
ab
+ 25.43
54.77±1.24
ab
+ 10.65
42.99±5.21
Annona mucosa
9.30±1.22
bc
+14.81
33.30±3.26
b
+ 7.77
44.20±3.56
bc
+ 9.16
53.40±1.97
ab
+ 7.88
41.91±7.32
Annona muricata
7.30±0.20 c
- 9.87
29.00±0.83
bc
- 6.15
43.00±2.05
bc
+ 6.17
53.80±1.07
ab
+ 8.69
42.23±3.94
Annona sylvatica
4.80±0.23
d
- 40.74
25.40±0.91
c
- 17.80
38.50±1.98
c
- 4.94
52.00±1.71
ab
+ 5.05
40.82±6.77
Control (acetone:water, 1:3
(v/v))
8.10±0.83
bc
--
30.90±2.71
bc
--
40.50±2.85
c
--
49.50±2.66
b
--
38.85±5.11
Negative control (water)
15.60±0.25
a
--
46.30±1.29 a
--
54.10±1.34
a
--
55.95±0.05
a
--
43.92±4.49
F
37.662
18.21
8.0269
2.4677
0.6521ns
P value
<0.0001
<0.0001
<0.0001
0.04378
0.6439
1 Means followed by different letters, in the columns, indicate significant differences between treatments
(GLM with Gaussian distribution followed by Tukey’s post hoc test, P<0.05)
2 P.I.: Percentage of inhibition;
ns: Not significant (P>0.05).
DISCUSSION
Our study provides important information regarding
promising sources of compounds to be used as grain
protectors in the preventative management of
Coleoptera pest species in stored cereals. Annonaceae
is one of the most diverse and abundant plant family
in Neotropical forests (Chatrou et al., 2004; Maas et
al., 2011a; Maas et al., 2011b) and has been shown to
exhibit secondary metabolites with great chemical
diversity and promising biological activities (Lebouef
et al., 1982; Zafra-Polo et al., 1998; Colom et al.,
2010); however, this study represents the most
comprehensive screening study performed to date.
Despite the variations in soil-climate
conditions in the sampling sites, which could have
influenced the chemical profiles of the extracts, and
the differences in the sampling effort for the different
plant structures, genus Annona seeds were identified
as the main sites of accumulation of compounds with
activity against insects. Therefore, our results are
consistent with other reports in the literature
(Leatemia & Isman, 2004; Llanos et al., 2008; Seffrin
et al., 2010; Grzybowski et al., 2013; Ribeiro et al.,
2013). Concerning the action on insects, this study is
the first to report the activity of compounds derived
from A. sylvatica (formerly Rollinia sylvatica) on
pests associated with stored grains. To date, a small
number of secondary compounds from A. sylvatica,
native to the center-south region of Brazil (Lorenzi et
al., 2005), were isolated and evaluated for their
bioactive potential. Consistent with our results,
Mikolajczak et al. (1990) demonstrated the oral
toxicity of a hexanic extract from A. sylvatica fruits
against Ostrinia nubilalis (Hübner) (Lepidoptera:
Crambidae) larvae. After successive fractionation, the
compound sylvaticin (the only acetogenin reported
from this species to date) was isolated and shown to
exhibit a series of biological activities, including the
protection of cantaloupe plants against Acalymma

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/228
vittata Barber (Coleoptera: Chrysomelidae).
Recently, Formagio et al. (2013) demonstrated the
anti-inflammatory and anti-carcinogenic properties of
essential oils of A. sylvatica leaves, which are mostly
composed of a combination of oxygenated
sesquiterpenes.
A number of reports in the literature ratify the
putative toxicological effects (acute or chronic) of
derivative compounds from the remaining species of
Annona for medically and agriculturally relevant
pests. Therefore, compounds from A. muricata
demonstrate activities against Plutella xylostella
(Linnaeus) (Lepidoptera: Plutellidae) (Trindade et al.,
2011), Bactericera cockerelli (Sulc) (Hemiptera:
Triozidae) (Flores-Davila et al., 2011), Anastrepha
ludens (Loew) (Diptera: Tephritidae) (Gonzalez-
Esquinca et al., 2012) and Aedes aegypti (Linnaeus)
(Diptera: Culicidae) (Grzybowski et al., 2013).
Acetogenins isolated from A. montana seeds
demonstrate toxicity for Oncopeltus fasciatus
(Dallas) (Hemiptera: Lygaeidae) (Colom et al., 2008)
and insecticidal and anti-feeding activities for
Spodoptera frugiperda (J.E. Smith) (Lepidoptera:
Noctuidae) larvae (Blessing et al., 2010).
Furthermore, the extract obtained through the
decoction of A. mucosa seeds has a repellent effect on
Acromyrmex octospinosus (Forel) (Hymenoptera:
Formicidae) workers (Boulogne et al., 2012).
Using S. zeamais as a model, Llanos et al.
(2008) demonstrated the efficient control of adults
and the complete inhibition of the F1 progeny using
extracts (at concentrations higher than 2,500 ppm)
from A. muricata seeds using hexane and ethyl
acetate as solvents. In previous studies, we
demonstrated that A. montana (Ribeiro, 2010) and A.
mucosa (Ribeiro et al., 2013) seed extracts in both
hexane and dichloromethane solvents caused
promising bioactive effects on S. zeamais. In contrast
to the solvents used in this study, the extractions in
the previous study were performed using organic
solvents at gradients of increasing polarity in all
cases. Given the changes in the chemical profiles of
the derivatives obtained using different techniques
and/or solvents, this difference can partially explain
the differences in bioactive concentrations observed
between the studies. Meanwhile, this study
demonstrates the possibility of extracting active
principles of these species using a “green solvent”
that is naturally biodegradable and produced from
renewable sources.
A large number of compounds of diverse
chemical natures in several structures of the genus
Annona have been isolated in a number of
phytochemical studies (Lebouef et al., 1982; Chang
et al., 1998; Kotkar et al. 2001). Among the
compounds, acetogenins stand out because of their
structural abundance and the wide array of biological
activities they exhibit, such as powerful insecticidal
and acaricidal activities (Alali et al., 1999; Colom et
al., 2008, 2010). Acetogenins are potent inhibitors of
complex I (NADH:ubiquinone oxidoreductase) of the
mitochondrial electron-transport system and of the
enzyme NADH:oxidase in the cell membrane of
target arthropods (Lewis et al., 1993). According to
Bermejo et al. (2005), the majority of acetogenins in
Annonaceae have been isolated from the seeds and
stems of the genera Annona, Anomianthus, Asimina,
Desepalum, Goniothalamus, Rollinia [now Annona
(Rainer, 2007)], Polyalthia, Porcelia, Uvaria and
Xylopia. However, the relative content of the
derivatives originating from the different genera and
the structure-activity relationship of the acetogenins
from different structures remain to be investigated.
Based on the estimated lethal time (in the
LC90 estimated for each selected extract), the slower
action of the active compounds was confirmed. The
symptomatology of the contaminated insects was
characterized by the inactivity, locomotive instability
and food avoidance, followed by the collapse,
paralysis, and slow death by respiratory insufficiency.
These signs are typical of the action of compounds
that inhibit mitochondrial respiration, such as
rotenone and piericidin (Ware and Whitacre, 2004.).
Therefore, based on these findings and previous
analysis (Ribeiro et al., 2013), it is possible to
hypothesize that the biological activity of the extracts
from A. sylvatica seeds and A. montana, A. mucosa
and A. muricata seeds and leaves are because of the
presence of acetogenins.
Despite the effects were less expressive
compared with those observed for the extracts of the
genus Annona, this study is the first to report the
activity of D. lanceolata derivatives on pest insects, a
plant species that has not been studied from a
phytochemical point of view. However, a number of
pharmacological properties, such as antiprotozoal
(Tempone et al., 2005), antinociceptive and anti-

Ribeiro et al.
Searching for sources of grain protectors in Neotropical Annonaceae
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/229
inflammatory activities (Sousa et al., 2008), of crude
ethanolic extracts and/or the isoquinoline alkaloid-
rich fraction from D. lanceolata leaves have been
reported in the literature and corroborate the potential
observed in this study.
To better elucidate the potential protectant
effect on grains, the antifungal and antiaflatoxigenic
activities of the most promising extracts (regarding
the action on S. zeamais) were evaluated using the A.
flavus isolate. Although the fungal toxicity of the
crude extracts and acetogenins isolated from the
genus Annona species were reported previously for a
number of plant species (Dang et al., 2011) and
human pathogens (Ahmad and Sultana, 2003; Lima et
al., 2011), the extracts evaluated did not exert
pronounced effects on A. flavus. Hypothetically, the
lack of fungicidal effect could be because of
evolutionary selection in A. flavus species adapted to
coexist with these secondary metabolites.
Corroborating this hypothesis, Okwulehie and Alfred
(2010) demonstrated that A. flavus is a species that
deteriorates A. muricata fruits in Nigeria. However,
because the insects are the main agent of dispersion
of fungal spores in the grain mass, the adequate
control of pest insect species provided by the
respective extracts can decrease the incidence of A.
flavus and consequently the aflatoxin levels in the
stored grains.
Despite the preliminary nature of the data in
this study, it can be concluded that ethanolic extracts
from A. sylvatica seeds, D. lanceolata leaves and A.
montana, A. mucosa and A. muricata seeds and
leaves exert bioactive effects on S. zeamais.
Accordingly, bio-guided studies are being conducted
in order to purify, isolate and characterize the
compounds responsible for the observed bioactivity.
Additionally, it will be possible to evaluate the
potential use of these compounds as model-molecules
or biorational compounds in integrated management
programs of the stored pests. However, this study
provides a scientific basis for the rational utilization
of Annonaceae species with potential use to humans,
mainly as a homemade tool for stored grain
protection.
ACKNOWLEDGMENTS
The authors thank Dr. Sérgio Sartori from the Rare
Fruits Brazilian Association (Rio Claro, SP, Brazil);
Dr. José Bettiol Neto from the Fruit Center of the
Agronomic Institute of Campinas (IAC/APTA,
Jundiaí, SP, Brazil) and Vale Natural Reserve
(Linhares, ES, Brazil), for the help with obtaining the
Annonaceae species used in this study. We also thank
the São Paulo Research Foundation (FAPESP, grant
number 2010/52638-0) and the National Science and
Technology Institute for Biorational Control of Pest
Insects (INCT-CBIP, grant number 573742/2008-1)
for the financial support.
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